5-Hydroxymethylcytosine (5-hmC) is a newly discovered base modification in mammalian genomic DNA. Recent studies strongly suggest that 5-hmC is another vital epigenetic mark that plays major and broad roles in gene regulation. Because the sequencing methods currently available cannot differentiate 5-meC from 5-hmC, we recently developed a chemical-labeling strategy for 5-hmC sequencing through which we have successfully obtained the first 5-hmC- distribution map in a mammalian genome. In this application, I propose to develop new single- base resolution sequencing methods to determine the precise locations of 5-hmC in genomic DNA, which will allow us to address the exact roles of 5-hmC in gene regulation. In addition, using the synthesized DNA oligos as models, we have found that the 5-hmC modification in DNA oligos could be oxidized to 5-formylcytosine (5-fC) and 5-carboxylcytosine (5-caC) by TET proteins in vitro; moreover, both 5-fC and 5-caC modifications could be detected from genomic DNA isolated from TET over-expressed cells. These findings suggest that 5-fC and 5-caC modifications are also present in the genomic DNA of certain cells. In this application, I propose to develop new methods for labeling and enriching these modifications for their detection and sequencing in genomic DNA. These methods, if successful, will lay the groundwork for the study of the potential roles of these modifications in gene regulation and development. During this project, I will make use of my expertise in nucleic acid chemistry and work closely with my mentor and collaborators to extend my knowledge and experimental skills in biochemistry, cell biology and genomics to address key questions related to 5-hmC, 5-fC, and 5-caC modifications in genomic DNA. This K01 award will not only allow me to study these fundamental questions, but also prepare me for an independent career in research in this area in the future.